The present disclosure relates to a labeled compound used for detection of a sample molecule and more particularly, to a labeled compound designed to permit an aromatic tertiary amine compound to be capable of binding with the sample molecule and also to a detection method using the same.
There are known techniques of analyzing biomolecules wherein sample molecules made of various types of biomolecules such as of nucleic acids, proteins, sugars, enzymes, hormones, antigens, antibodies and the like are fluorescently labeled and the fluorescence is detected. This analyzing technique is a fundamental one in the fields of the analysis of life phenomena, genetic diagnosis, tissue engineering and the like and is also an essential one in the screening of medical drugs. For instance, labeling of gene, protein with a fluorescent dye enables a specific type of gene or protein in an analyte to be detected at high sensitivity.
Comprehensive analyses of analytes have been conducted, for example, by labeling many types of oligonucleotides or peptides with fluorescent dyes and using DNA chips or protein chips fixing a microarray of the labeled oligonucleotides or peptides thereon. Moreover, in an immunostaining or molecule imaging technique wherein a specific type of biomolecule such as, for example, of a sugar, protein or the like is labeled with a fluorescent dye and is observed through a fluorescence microscope after generation of a fluorescence, dynamic analyses of an intracellular or intercellular molecule become possible.
The labeled probe obtained by labeling DNA, a DNA base derivative, a DNA oligonucleotide or the like with a fluorescent dye is employed in northern blotting, Southern blotting, in situ hybridization, DNA sequence determination reaction and the like. The antibody or antigen labeled with a fluorescent dye is used for immunoassay making use of an antigen-antibody reaction. There is also known an enzyme immunoassay method to which the avidin (streptoavidin)-biotin linkage is applied and in which avidin modified with a fluorescent dye is used.
For the labeling of a biomolecule serving as a sample to be detected, for example, an aromatic dye compound such as FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine isothiocyanate), fluorescein, rhodamine, coumarin, cyanine dye or the like is used as the fluorescent labeling reagent.
Techniques relating to the detection of biomolecules and various types of fluorescent labeling compounds used for the detection of biomolecules are known and reported as shown below.
In Japanese Patent Laid-open No. 2005-208026 (at Paragraphs 0004-0011), entitled “Method of detecting biological molecules, and labeling dye and labeling kit used for the same,” (referred to as Patent Document 1) there is the following description.
The method for detecting a biomolecule in the Patent Document 1 is characterized in that a biomolecule sample and an organic EL (electroluminescence) dye are reacted with each other and the fluorescence of the biomolecule sample labeled with the organic EL dye is measured. It is stated that the organic EL dye used is made of a compound containing a conjugated five-membered ring compound, for which there can be used a compound containing at least one hetero atom, selenium atom or boron atom. Moreover, there may be further used a condensed polycyclic compound made of a five-membered ring compound and a conjugated six-membered ring compound, wherein the five-membered ring compound used may be an azole derivative or an imidazole derivative.
Prior to the reaction with a biomolecule, the organic EL dye may be introduced thereinto with any one of functional groups selected from an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group and an active-esterified carbonyl group.
The labeling dye of the Patent Document 1 is one used for detection of a biomolecule based on fluorescent measurement and is characterized in that it consists of an organic EL dye having a reactive group capable of binding to the biomolecule. It is stated that as the reactive group, there can be used any one of functional groups selected from a carboxylic group, an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group and an active-esterified carbonyl group.
It is also stated in the Patent Document 1 that the organic EL dye is a compound containing a conjugated five-membered ring compound and a compound containing at least one hetero atom, selenium atom or boron atom may be used as this five-membered ring compound. In addition, there may be further used a condensed polycyclic compound composed of the five-membered ring compound and a conjugated six-membered ring compound. The five-membered ring compound may be an azole derivative or an imidazole derivative.
A variety of organic EL dyes are known aside from those set out in the Patent Document 1. For instance, there have been reported organic EL dyes composed of styryl compounds having a diversity of structures, for example, in Patent Documents indicated below:                Japanese Patent No. 3852517 (paragraphs 0017 to 0020),        Japanese Patent No. 3852518 (paragraphs 0017 to 0020),        Japanese Patent No. 3852520 (paragraphs 0015 to 0018),        Japanese Patent No. 3555736 (paragraphs 0017 to 0020),        Japanese Patent No. 3820752 (paragraphs 0017 to 0020),        Japanese Patent Laid-open No. 2000-230132 (paragraphs 0008 to 0021),        Japanese Patent Laid-open No. 2001-106657 (paragraphs 0008 to 0019),        Japanese Patent Laid-open No. 2001-106658 (paragraphs 0008 to 0012),        Japanese Patent Specification No. 3852552 (paragraphs 0017 to 0037),        Japanese Patent Laid-open No. 2001-131128 (paragraphs 0008 to 0012),        Japanese Patent Laid-open No. 2001-288377 (paragraphs 0007 to 0020),        Japanese Patent Laid-open No. 2002-226722 (paragraphs 0007 to 0014),        Japanese Patent Laid-open No. 2004-87463 (paragraphs 0021 to 0026),        Japanese Patent No. 4001118 (paragraphs 0013 to 0065),        Japanese Patent Laid-open No. 2005-5226 (paragraphs 0013 to 0057),        Japanese Patent Laid-open No. 2005-35927 (paragraphs 0013 to 0042),        Japanese Patent Laid-open No. 2006-124333 (paragraphs 0011 to 0054),        Japanese Patent Laid-open No. 2006-128437 (paragraphs 0009 to 0024), and        Japanese Patent Laid-open No. 2006-273737 (paragraphs 0009-0027).        